Purification of the Rv0045c protein by ion exchange chromatography and gel filtration chromatography.

<p>The Rv0045c protein was purified by anion exchange chromatography (A), cation exchange chromatography (B), and gel filtration chromatography (C). The purity was checked by SDS-PAGE analysis after each purification procedure.</p

CD spectra of the Rv0045c protein at different pH and temperatures.

<p>The CD measurements were made in the presence of various pH (A) at pH 2.0 (black), pH 3.0 (red), pH 4.0 (yellow), pH 6.0 (blue), pH 7.0 (purple), pH 8.0 (pink), pH 9.0 (green), pH 10.0 (gray), pH 11.0 (coral) and pH 12.0 (light green) at room temperature, and different temperatures (B) at 10°C (black), 20°C (gray), 30°C (yellow), 40°C (green), 50°C (blue), 60°C (purple) and 70°C (red) at pH 7.5, respectively. Values represent the mean ± SD of three analyses. The concentration of the Rv0045c protein was fixed at 0.35 mg/mL (20 mM Tris, pH 7.5).</p

SDS-PAGE analysis for expression and affinity chromatography of the Rv0045c protein.

<p>Lane 1, culture pellet (uninduced); Lane 2, culture pellet (induced with 0.3 mM IPTG at 16°C); Lane 3, the supernatant of induced cells after sonication; Lane 4, fluid through Ni²⁺-affinity chromatography column; Lane 5 and 7: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 200 mM Imidazole, pH 7.5; Lane 6 and 8: purified Rv0045c protein eluted by 20 mM Tris, 150 mM NaCl, 500 mM Imidazole, pH 7.5; Lane M: molecular mass markers.</p

MALDI-TOF peptide mass fingerprint (PMF) spectrometry of the Rv0045c protein.

<p>The PMF analysis was made from fragments of purified Rv0045c protein derived through trypsin digestion. The expected tryptic masses clearly matched, with 1 Da tolerance, the calculated values. The sequence coverage of these fragments was shown in bold red.</p

Relative enzyme activity of the Rv0045c protein toward <i>p</i>-nitrophenyl derivatives at pH 7.0 and 37°C.

<p><i>ND</i>: Not detectable</p>a<p>The specific activity toward <i>p</i>-nitrophenyl caproate (C₆) corresponding to 3.5 U/mg protein/min was defined as 100%. And one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min.</p

Effects of temperature and pH on enzyme activity of the Rv0045c protein.

<p>The enzyme activities were measured using <i>p-butyrate caprylate (C₆)</i> as substrate in the presence of mild temperatures (36°C–40°C) at pH 6.0 (red), pH 7.0 (green) and pH 8.0 (blue). Values represent the mean ± SD of five analyses. The concentration of the Rvoo45c protein was fixed at 0.2 mg/mL (20 mM Tris, pH 7.5). The enzyme activities were expressed as units hydrolase/mg protein/min (one hydrolase unit is the quantity of enzyme required to increase absorbance by 0.01 units at 405 nm per min).</p

Enantioselective Bromolactonization of Deactivated Olefinic Acids

A novel enantioselective bromolactonization of α,β-unsaturated ketones using bifunctional amino-urea catalysts has been developed. The scope of the reaction is evidenced by 23 examples of halolactones bearing various functionalities with up to 99% yield and 99:1 er. Unlike typical urea catalysts that require electron-deficient substituents to enhance the hydrogen bond strength, it is interesting to realize that electron-rich ureas are essential for high enantioselectivity in this case. Moreover, experimental data reveals that the halolactone compounds exhibit considerable anti-inflammatory effects on LPS-induced RAW 264.7 cells

Proposed mechanisms for the hydrolysis of <i>p</i>-nitrophenyl caproate.

<p>(A) The reaction for the hydrolysis of <i>p</i>-nitrophenyl caproate by Rv0045c. Rv0045c hydrolyzes <i>p</i>-nitrophenyl caproate to produce <i>p</i>-nitrophenol and caproic acid. (B) Proposed mechanisms for the hydrolysis reaction. Mechanism 1 utilizes Ser154 to activate a water molecule for attacking the carbonyl carbon of the C-O ester bond. Mechanism 2 utilizes Ser154 to directly attack the carbonyl carbon of the C-O ester bond.</p

Sequence alignment of Rv0045c with other structurally homologous enzymes.

<p>Included enzymes are E-2AMS hydrolase (PDB ID: 3KXP), methylesterase PME-1 (PDB ID: 3C5V), hydrolase YP_496220.1 (PDB ID: 3BWX), CarC enzyme (PDB ID: 1J1I), esterase ybfF (PDB ID: 3BF7) and soluble epoxide hydrolase (PDB ID: 1EHY). Secondary structural elements and every the tenth residue of Rv0045c are indicated above the alignment. The “nucleophile elbow” of G-X-S-X-G sequence motif is marked with green stars and the nucleophilic serine residue Ser with a blue circle. The other catalytic residue is labeled using a purple triangle. Strictly conserved residues with the identity of >80% are highlighted by pink front.</p

Overall structure of Rv0045c.

<p>(A–B) Cartoon representation of Rv0045c in two views related by a vertical rotation of 90 degrees. The secondary structural elements (α1–α12, β1–β10) were labeled. The core structural elements are colored slate and the cap domain in violet (A). α-helices (red) and β-strands (yellow) are differentiated by colors (B). (C) Secondary structure diagram of the α/β fold core of Rv0045c. α-helices, β-strands and the cap domain are represented by red cylinders, yellow arrows and a violet ellipse, respectively. The α/β fold core consists of a mostly parallel, 8-stranded β sheet surrounded on both sides by α-helices (only β2 is antiparallel). The nucleophilic residue, Ser154, positioned at the beginning of α5, is marked with a black dot. (D) Topology diagram of Rv0045c using the same color scheme as (B). The missing region between α6 and α7 (residues 194–204) is represented as dotted line.</p